High dose radionuclide complexes for bone marrow suppression

ABSTRACT

The present invention relates to a method of suppressing bone marrow (BM) and treating conditions that arise in or near bone such as cancer, myeloproliferative diseases, autoimmune diseases, infectious diseases, metabolic diseases or genetic diseases, with compositions having as their active ingredient a radionuclide complexed with a chelating agent such as macrocyclic aminophosphonic acid.

RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 10/882,054,filed Jun. 30, 2004, entitled HIGH DOES RADIONUCLIDE COMPLEXES FOR BONEMARROW SUPPRESSION, which is a divisional of U.S. patent applicationSer. No. 10/784,476, filed Feb. 23, 2004, entitled HIGH DOSERADIONUCLIDE COMPLEXES FOR BONE MARROW SUPPRESSION, which is acontinuation application of U.S. patent application Ser. No. 10/159,245,filed May 29, 2002, entitled HIGH DOSE RADIONUCLIDE COMPLEXES FOR BONEMARROW SUPPRESSION, which is a divisional application of U.S. patentapplication Ser. No. 10/014,335, filed Dec. 11, 2001, Now U.S. Pat. No.6,767,531, Issued Jul. 27, 2004, entitled HIGH DOSE RADIONUCLIDECOMPLEXES FOR BONE MARROW SUPPRESSION, which is a continuation under 35USC § 111(a) of PCT Application Serial No. PCT/US00/16052, filed on Jun.12, 2000 and published as WO 00/76556 on Dec. 21, 2000, which claimspriority from provisional U.S. Patent Application No. 60/139,065, filedJun. 11, 1999, 60/143,780, filed Jul. 13, 1999 and 60/149,821, filedAug. 19, 1999, all of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The use of agents which cause partial or total suppression oreradication of bone marrow has become an accepted part of certainprocedures used to treat patients with cancers such as leukemias,lymphomas, myelomas and Hodgkin's disease as well as in the treatment ofpatients suffering from hematopoietic disorders such as sickle cellanemia and thalassemia. In situations where the patient is sufferingfrom a hematopoietic disorder such as thalassemia or sickle cell anemia,bone marrow transplantation may offer the possibility of a cure. If theabnormal bone marrow of an individual suffering from sickle cell anemiaor thalassemia can be eradicated and then replaced with a bone marrowthat takes and is reproduced and capable of producing normal red cellswith normal hemoglobin, the individual may be cured.

Multiple myeloma is a disease of abnormal plasma cell proliferation thatcan result in anemia, pathologic fractures, renal failure, and death.Complete eradication of the abnormal plasma cells and precursor abnormalcells that may differentiate into abnormal plasma cells can prevent theprogression, reverse or even cure the disease.

Current therapy is high dose chemotherapy (melphalan or combinationssuch as thiotepa/busulfan/cyclophosphamide) with or without total bodyirradiation (TBI). Treatment with melphalan 140 mg/m² of body-surfacearea given intravenously can induce complete remissions in about 20-30%of patients. However, it causes severe and sometimes irreversiblemyelosuppression. For example, see B. Barlogie et al., Blood, 72, 2015(1989); (1998); D. Cunningham et al., J. Clin. Oncol., 12, 764 (1994);R. Bataille et al., New Engl. J. Med., 336, 1657 (1997).

Furthermore, when radiation is combined with other cytotoxic therapies,such as chemotherapy, the toxicity can be additive or synergistic. Inaddition, patients who undergo bone marrow suppression or ablation,sufficient to require either growth factor support, transfusion supportor stem cell reinfusion, may encounter toxicities from the chemotherapy,from TBI, or both.

The dose of chemotherapy and radiotherapy that can be administered to anindividual patient is often limited by patient age or overall health.Some patients who could benefit from high dose chemotherapy andradiotherapy do not receive it because they are considered to old orhave other concomitant diseases which make them unsuitable candidatesbecause of the non-target organ toxicity currently associated with thesetherapies. Higher doses of radiation may increase the percentage oftumor cells that are killed, and, with ionizing radiation, there comes apoint where small increments in radiation can have a major impact on thepercentage of cells killed.

The use of complexed radionuclides for bone marrow suppression isdiscussed in U.S. Pat. No. 4,853,209, where the use of Samarium-153(¹⁵³Sm), Gadolinium-159 (¹⁵⁹Gd), or Holmium-166 (166Ho) complexed with aligand selected from ethylenediaminetetramethylenephosphonic acid(EDTMP), diethylenetriaminepentamethylenephosphonic acid (DTPMP),hydroxyethylethylenediaminetrimethylenephosphonic acid (HEEDTMP),nitrilotrimethylenephosphonic acid (NTMP), ortris(2-aminoethyl)aminehexamethylenephosphonic acid (TTHMP) isdisclosed. Phosphonic acid-containing chelators are selected due totheir ability to target the radionuclide to the bone.

U.S. Pat. Nos. 4,882,142, and 5,059,412 are directed to a method for thesuppression of bone marrow and to a composition for use in the method.The method comprises administering to a mammal in need of such treatmenta bone marrow suppressing amount of at least one composition comprisedof a radionuclide ¹⁵³Sm, ¹⁵⁹Gd, or ¹⁶⁶Ho complexed with1,4,7,10-tetraazacyclododecanemethylenephosphonic acid as themacrocyclic chelating moiety. The method of bone marrow suppressiondescribed therein may be used in combination with chemotherapeutic drugsand/or external radiation. The compositions comprise the radionuclidesin dosages comprising from about 18 to 1850 megabecquerels per kilogramof body weight of the target mammal. The amount of radioactivitydelivered to the bone is necessarily lower, and was not determined.

Therefore, a continuing need exists for methodologies and agents usefulfor selective bone marrow suppression and/or for adequate tumor cellkilling, that is, wherein the bone marrow is suppressed and/or tumorcells killed with only minimal damage to non-target soft tissues, forexample, liver, urinary bladder, and kidney. There is also a need for ameans of delivering high radiation doses to sites of disease in or nearbone, with standard or high dose chemotherapy without increasingtoxicity to non-target organs. For those situations where bone marrowsupport can aid in therapy or cure, it would be desirable to have ameans of first selectively suppressing the abnormal or diseased bonemarrow independent of, or with limited, total body irradiation.

SUMMARY OF THE INVENTION

The present invention provides a method for selectively, rapidly, andeffectively suppressing bone marrow or to treat a pathology associatedwith (in or near) the bone or bone marrow. In one aspect, the methodcomprises administering to a mammal in need of such treatment a highdosage of a complex of a bone marrow suppressing radionuclide with abone targeting ligand, such as an aminophosphonic acid. Such pathologiesinclude cancer, autoimmune diseases, certain infections and certainhematopoietic genetic disorders.

Preferably, the radionuclide is ¹⁶⁶Ho and the ligand is a macrocyclicaminophosphonic acid such as DOTMP. The complex is preferablyadministered in a single treatment dose effective to deliver at least 20Gy to the bone marrow of the subject. The present invention alsoprovides aqueous compositions comprising ¹⁶⁶Ho-DOTMP and aradioprotectant that are stable for at least about 72 hours underambient conditions.

A preferred embodiment of the invention provides a method to increasethe efficacy of chemotherapy, particularly high dose or intensivechemotherapy, without a substantial increase in total side effects, andmore preferably, without the need for TBI. This method comprisesadministering an effective bone marrow suppressing amount of aradionuclide-amino phosphonate complex to a subject in need of suchtreatment in conjunction with one or more chemotherapeutic agents, whilemaintaining an acceptable level of tolerance of the subject to the totaltherapeutic regimen. For example, it has been unexpectedly found that ahigh dosage of radiation can be delivered to the bone marrow of asubject afflicted with a bone-associated neoplasm (cancer) ornon-cancerous myeloproliferative disorder in conjunction with high dosechemotherapy, such as melphalan in the case of myeloma, while notsubstantially increasing the side effects as compared to the sideeffects associated with the high dose chemotherapy alone.

For example, the use of at least about 200 mg/m² melphalan to treatmultiple myeloma can be combined with a dosage of a ¹⁶⁶Hoaminophosphonate complex effective to deliver about 20-60 Gy, preferablyabout 30-50 Gy, to the bone marrow of the afflicted subject withoutsubstantially increasing the side effects over those associated withmelphalan therapy alone at about 140 mg/m² or about 200 mg/m². Suchtreatment has the advantage of providing efficacy comparable to thatobtained from treatment with a combination of melphalan and TBI, withoutthe side effects associated with TBI.

The efficacy of conventional melphalan therapy (i.e., 70-120 mg/m² canalso be enhanced by administration of the present complexes, thusimproving the outcome for older patients. Therefore, the efficacy ofcurrent treatment regimens to treat multiple myeloma, e.g., 140 mg/m²melphalan plus TBI or 200 mg/m² melphalan alone, can be substantiallyenhanced without substantial increase in side effects, e.g., those dueto melphalan and/or TBI used without the complex.

The preferred radionuclide compositions employed in the method of thepresent invention are capable of delivering a significant portion,preferably greater than about 15%, e.g., about 25-35% of theradioactivity present in the composition to bone tissue while notdeleteriously affecting non-target soft tissues. Therefore, for thosedisease states where the treatment regimen requires bone marrowsuppression, the present invention is particularly advantageous since itprovides a means of achieving selective reduction in the hemopoieticcell population, without having to resort to external irradiation of thesubject, e.g., to TBI, resulting in minimal damage to non-targettissues. The reduction in the radiation dose delivered to non-targettissues (as compared to the use of TBI alone), provides the opportunityto use the same or increased amounts of conventional chemotherapeuticregimens, particularly non-radioactive antineoplastic (“anti-cancer”)agents that per se suppress bone marrow, such as alkylating agents.

It may be possible to completely eliminate the use of targeted radiationor TBI in certain patient populations, such as those under 55 years ofage, while retaining equivalent efficacy. It may also be possible toincrease the efficacy of regimens in which TBI is desirable, but toohazardous to use, as in older patients (>55 years of age). However, ifit is desirable to employ targeted irradiation or TBI in conjunctionwith the bone marrow suppression method described herein, for example,in the treatment of leukemia, it can be possible to reduce the radiationdosage used for the total body irradiation and still obtain the same orhigher level of reduction of leukemic cells.

Preferred radionuclide complexes comprise radionuclides that exhibithalf-lives of sufficient length so that they can deliver preselectedhigh doses of radiation after bone-targeting and soft tissue clearance,but which exhibit half-lives sufficiently short so that they decay in arelatively short time to allow safe bone marrow or stem celltransplantation or other therapy. For example, ¹⁶⁶Ho has an energeticbeta-particle with a long path length. Yet, despite increasing the doseof ¹⁶⁶Ho from about 20 Gy to about 50 Gy to the marrow along withmoderately high or very high doses of chemotherapy, there has beensurprisingly no increase in toxicity to other organs beyond thatexpected from the chemotherapy itself and, surprisingly, no evidence ofdelay or difficulty in engraftment of marrow or stem cell transplant dueto direct toxicity to the bone marrow space. The rapid radioactive decayalso unexpectedly pennits subsequent use of high dose chemotherapy,since cumulative effects are avoided or lessened. Thus, the presentmethod provides the basis for a potent combination therapy, particularlywith respect to cancers that are associated with bone, because additivetoxic side effects are readily avoided.

In one aspect of the invention, the complex of the macrocyclicaminophosphonic acid, 1,4,7,10-tetraazacyclododecane, and ¹⁶⁶Ho wasfound to deliver higher doses of radiation to the bone or to adjacentareas than previously thought possible, without undue deleterious sideeffects. A preferred ratio of DOTMP to ¹⁶⁶Ho is above 3; preferablyabout 3.5-5, most preferably about 3.5.

Furthermore, it was unexpectedly found that bone marrow can be ablatedeffectively with a single dose or with closely spaced dosing regimens,further reducing the handler's exposure to radiation. As used herein,the term “single dosage” or “single dose” means that the total dosage ofradionuclide complex is administered in one (1) or more doses within ashort period of time, e.g., less than about 24 hours. Preferably thedoses will be administered within about 12 hours, more preferably withinabout 8 hours. Most preferably the doses will be administered withinabout 0.1-4 hours. Preferably the dose will be also administered as asingle infusion or injection.

Preferably, an effective bone marrow suppressing dose of a radionuclideaminophosphonic acid complex, such as ¹⁶⁶Ho-DOTMP will administer atotal dose of 20-60 Gy, preferably about 30-50 Gy and, most preferably,about 37-45 Gy of radiation to the bone/bone marrow of the subject. Atabout 30% uptake, e.g., for a human subject, total therapy dose to bonemarrow is about 500-4000 mCi (about 18.5-148 GBq).

Because the actual percentage of the administered dose of radiation thatreaches the bone/bone marrow necessarily varies from subject to subject,the present method also preferably comprises the steps of firstadministering a dose (the “diagnostic or dosimetry dose”) of aradionuclide complex effective to determine the dosage required tosubsequently deliver an effective therapy dose or doses, and thendetermining the percent uptake of the diagnostic or dosimetry dose bythe bone of the subject, e.g., via whole body retention measurements.Although a radionuclide other than the intended therapeutic radionuclidecan be used for dosimetry measurements, it is preferable to use the sameradionuclide for dosimetry measurements and for therapy.

The administered dosage can, in some cases where patients haverelatively low uptake in the skeleton, contain from about 2000 to about2750 megabecquerels (MBq) per kilogram of body weight of said mammal.The most preferred dosage contains from about 2000 to about 2500megabecquerels per kilogram of body weight of said mammal.

The dosing is preferably accomplished with a radionuclide complexemitting a beta energy of >0.5 MeV and having a radionuclide half-lifeof less than 5 days, most preferably <3 days, at a beta energy of >1MeV. Preferred radionuclides include radionuclides selected from thegroup consisting of ¹⁵³Sm, ⁹⁰Y, ¹⁵⁹Gd, ¹⁸⁶Re, ¹⁸⁸Re, and ¹⁶⁶Ho(half-life 26.8 hr.) complexed with a bone targeting complexing ligand.

The radionuclide complexes can be administered alone or in combinationwith adjuvant bioactive agents, that act in conjunction with thelocalized complex in order to treat diseases, such as disease orpathologies associated with (at or near) mammalian bone (including bonemarrow and associated tissue or cells). Such agents includeantineoplastic chemotherapeutic agents known to the art. The complex canbe delivered at a dose that itself is effective without the use of achemotherapeutic agent or irradiation from an external source. Suchregimens are particularly effective to treat cancers such as leukemia,myeloma, metastatic breast or metastatic prostate cancer, Hodgkin'slymphoma, osteosarcoma, Ewing's sarcoma or Paget's disease.

Following treatment with an amount of the present complexes, and,optionally, with external irradiation, growth factor support,chemotherapy, hormone therapy, or immunosuppressive therapy, thesubject's bone marrow can be augmented by blood marrow restoration, orregenerated, as by transplantation with purged autologous or matchedallogeneic bone marrow (including peripheral blood stem cells), and/orby treatment with bone marrow-stimulating agents.

The preferred chelating agents useful for practicing the presentinvention are polyaminophosphonic acid chelators, such as, for example,ethylenediaminetetramethylenephosphonic acid (EDTMP),diethylenetriaminepentamethylenephosphonic acid (DTPMP),hydroxyethylethylenediaminetrimethylenephosphonic acid (HEEDTMP),nitrilotrimethylenephosphonic acid (NTMP),1,4,7,10-tetraazacyclododecanetetramethylenephosphonic acid (DOTMP),tris(2-aminoethyl)aminehexamethylenephosphonic acid (TTHMP),1-carboxyethylenediamine-tetramethylenephosphonic acid (CEDTMP),hydroxyethylidene diphosphonate (HEDP),bis(aminoethylpiperazine)tetramethylenephosphonic acid (AEPTMP),N-methylethylenediaminetrimethylenephosphonic acid (MEDTMP),N-isopropylethylenediaminetriemthylenephosphonic acid (IEDTMP),N-benzylethylenediaminetrimethylenephosphonic acid (BzEDTMP), methylenediphosphonate, hydroxymethylene diphosphonate,ethane-1-hydroxy-1,1-diphosphonic acid, and the like. Other usefulchelating agents for radionuclides are generally disclosed in U.S. Pat.Nos. 5,059,412, 5,066,478, 5,300,279 and 4,897,254.

Preferred macrocyclic aminophosphonic acids are of the structure:

wherein substituents A, B, C, and D are independently selected fromhydrogen, hydrocarbon radicals having from 1-8 carbon atoms,

and physiologically acceptable salts of the acid radicals wherein X andY are independently selected from the group consisting of hydrogen,hydroxyl, carboxyl, phosphonic, and hydrocarbon radicals having from 1-8carbon atoms and physiologically acceptable salts of the acid radicals,and n is 1-3 with the proviso that when n>1, each X and Y may be thesame as or different from the X and Y of any other carbon atom; X′ andY′ are independently hydrogen, methyl, or ethyl radicals, and n′ is 2 or3, with the proviso that at least two of said nitrogen substituents is aphosphorus containing group, i.e., wherein N and P are connected byalkylene or substituted alkylene.

A more preferred macrocyclic aminophosphonic acid ligand is1,4,7,10-tetraazacyclododecanetetramethylenephosphonic acid (DOTMP).See, e.g., U.S. Pat. Nos. 4,973,333 and 5,714,604.

The present method can also be employed to treat pathologies other thancancer associated with (at or near) mammalian bone, that can beameliorated by partial bone marrow suppression or by complete bonemarrow ablation followed by bone marrow transplantation. The treatmentcan be accomplished by delivering i.e., 250-3000 megabecquerels per kgof body weight of the complex to the subject to be treated. Suchpathologies include, but are not limited to, immunological disorderssuch as autoimmune diseases, e.g., Crohn's disease, rheumatoid arthritisor multiple sclerosis; metabolic diseases, such as osteoporosis orosteopenia; infections and infectious disease such as tuberculosis orblastomycoses, inflammatory diseases such as osteomyelitis or Paget'sdisease; hematopoietic disorders, and conditions treatable with stemcell transplantation, with or without gene therapy, that utilize bonemarrow ablation, such as sickle cell anemia and lysosomal andperoxisomal storage diseases.

The present invention also provides novel liquid compositions,preferably aqueous compositions, comprising ¹⁶⁶Ho-DOTMP combined with aneffective stabilizing amount of ascorbic acid, gentisic acid, or otherradio-stable stabilizing agent buffered to pH 7-8, as well as methodsfor preparing the compositions. The ascorbic acid, gentisic acid, andthe like, maintain the radionuclide complex stability and reduces theamount of free radionuclide delivered in vivo. For example, ascorbicacid or gentisic acid may be present in the unit dosage forms useful inthe practice of the present invention at about 35-75 mg/ml ofcomposition. Stabilization unexpectedly inhibits radiolytic degradationof the complexes, i.e., so high (300 mCi/ml (12 GBq/ml)) levels of¹⁶⁶Ho-DOTMP can be maintained in the dosage forms, and thus allowsdistribution to hospitals at high levels of purity, with high levels of¹⁶⁶Ho-DOTMP.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graphical representation of the uptake of ¹⁶⁶Ho-DOTMP inbones and non-target organs.

FIGS. 2-4 are graphical representations of a comparison of the uptake of¹⁶⁶Ho-DOTMP in bones and non-target organs when using a stabilizer.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “mammal” means a warm blooded mammal, includinghumans, and is meant to encompass mammals in need of bone marrowsuppression, especially humans; thus in some instances the term“patient” or “subject” is alternatively used for mammal.

The term “disease” includes pathologies and deleterious conditions, suchas inflammatory responses, cancer, autoimmune, and genetic disorders.

The term “bone marrow restoration” includes partial or completeregeneration or augmentation of the bone marrow by marrowtransplantation or hematopoietic stem cell transplantation and/orstimulation of bone marrow regeneration by administration of growthfactors such as cytokines, glycoproteins and the like.

As used herein, the term “bone marrow transplant (BMT)” includesautologous, allogenic, xenogeneic marrow transplantation and stem celltransplantation.

The term “bone marrow suppression” refers to partial or essentiallytotal eradication (“ablation” or “myeloablation”) of the bone marrow, inparticular a temporary or permanent reduction of the hemopoietic stemcell population.

A sub-ablative therapy is one that does not completely eradicate bonemarrow, e.g., the marrow may recover, particularly if hematopoietic cellgrowth factors are administered.

As used herein, the term that external irradiation (targeted or TBI) isnot used “in conjunction with” the radionuclide complex and, optionally,chemotherapy, is intended to mean that external irradiation is notemployed as part of the same treatment protocol. For example, a patientcould have received external radiation treatment as part of a previoustreatment protocol and still be considered not to have received externalradiation “in conjunction with” treatment with the radionuclide complex.Thus, the term “inconjunction with” is intended to mean administrationas part of the same protocol radionuclide complex, in order toaccomplish the recited therapeutic effect, e.g., bone marrowsuppression.

As used herein, the term “substantial” when used with respect to theside effects of chemotherapy or radiation therapy is to be understood byreference to the art-recognized definitions and scales employed in theworking examples.

As used herein, the term “high dose” refers to a dose that is in theupper range of the dose used in conventional therapy to treat aparticular pathology, as recognized by the art. As defined in Example10, this can include the MTD ±10%. The dose range and highest typicaldose for certain chemotherapeutic agents is given herein below forillustration.

The present invention is directed to compositions and methods forsuppressing bone marrow and/or treating a disease in or near the bone orbone marrow that is ameliorated by said suppression. The presentinvention has significant benefits in that it permits rapid andselective bone marrow suppression (the bone marrow can be suppressedwith only minimal damage to non-target soft tissues, such as, forexample, lung, liver, stomach, mucosal linings and the like) without theneed for sustained exposure to radiation or for exposure to alarge, >about 15-20:1, molar ratio of chelating agent to radionuclide.The complexes of the invention can also be administered prophylaticallyor in an adjuvant setting with little evidence of disease but likelihoodof recurrence from minimal disease presence, e.g., to minimize theprobability of metastases of established cancer.

As will be more fully discussed later herein, the properties of theradionuclide, and of the radionuclide aminophosphonic acid complex areimportant considerations in determining which radionuclide compositionshould be employed for any particular treatment. For the purpose ofconvenience, the radionuclide aminophosphonic acid compositions willfrequently be referred to as “radionuclide complexes or compositions”and the aminophosphonic acid derivative referred to as the “ligand,”“chelator,” or “chelating agent”. The term “complexes” or “compositions”includes both the free compounds and the pharmaceutically acceptablesalts thereof.

Radionuclides

It is important that the half-life of the complexed radionuclides besufficiently long to allow for localization and delivery of the complexin the bone tissue via binding to chelator while still retainingsufficient radioactivity to accomplish essentially total bone marrowsuppression or eradication. The half-life also should be relativelyshort, so that after bone marrow irradiation is achieved, it is possibleto proceed with bone marrow or stem cell transplantation with minimaldelay prior to transplant, and in order to enhance the prospects of bonemarrow engraftment and patient recovery. Generally, it is preferred touse a radionuclide complex that results in rapid biolocalization of theradionuclide in the bone tissue so as to achieve rapid onset of bonemarrow irradiation. It is also beneficial to use a radionuclide havingsufficient beta energy, such that substantially all bone marrow cellsreceive a toxic irradiation from the targeted bone surfaces.

For example, radionuclides useful for bone marrow ablation can exhibitbeta energy >0.5 MeV, preferably >1 MeV with an effective half-life ofabout <5 days, preferably <3 days. Certain radionuclides such as Sr-89have been demonstrated, when selectively deposited in bone, to suppressbone marrow. [See, for example, Y. Shibata et al., J. Leukocyte Biol.,38, 659 (1985).] However, this compound is not clinically useful forthis purpose since the long half-life of Sr-89 (50 days) preventstransplantation of the new marrow for an unacceptable period of time.Radionuclides useful in the method and compositions of this inventionare Arsenic-77 (⁷⁷As), Molybdenum-99 (⁹⁹Mo), Rhodium-105 (¹⁰⁵Rh),Lutetium-177 (¹⁷⁷Lu), Cadmium-115 (¹¹⁵Cd), Antimony-122 (¹²²Sb),Promethium-149 (¹⁴⁹Pr), Osmium-193 (¹⁹³OS), Gold-198 (¹⁹⁸Au),Thorium-200 (²⁰⁰Th); preferably Samarium-153 (¹⁵³Sm), Yttrium-90 (⁹⁰Y),Gadolinium-159 (¹⁵⁹Gd), Rhenium-186 (¹⁸⁶Re), Rhenium-188 (¹⁸⁸Re), andHolmium-166 (¹⁶⁶Ho). Especially preferred is ¹⁶⁶Ho, which emits highenergy beta particles and gamma radiation (80 KeV, 6.0%) useful forimaging and exhibits a half-life of 26.8 hr.

The respective radionuclides can be obtained using procedures well knownin the art. Typically, the desired radionuclide can be prepared byirradiating an appropriate target, such as a metal, metal oxide, orsalt. Another method of obtaining radionuclides is by bombardingnuclides with particles in a linear accelerator or cyclotron. Yetanother way of obtaining radionuclides is to isolate them from fissionproduct mixtures. The method of obtaining the radionuclide is notcritical.

Chelating Agents

Aminophosphonic acids, particularly macrocyclic aminophosphonic acids,are the ligands of choice as chelators for the radionuclides useful inthe present methods. These compounds can be prepared by a number ofknown synthetic techniques. Generally, a compound containing at leastone reactive amine hydrogen is reacted with a carbonyl compound(aldehyde or ketone) and a phosphorous acid or appropriate derivativethereof.

Methods for carboxyalkylating macrocyclic amines to give aminederivatives containing a carboxylalkyl group are disclosed in U.S. Pat.No. 3,726,912. Methods to prepare alkylphosphonic acid amines andhydroxyalkylamines are disclosed in U.S. Pat. No. 3,398,198. See also,U.S. Pat. Nos. 5,066,478 and 5,300,279.

The amine precursor (1,4,7,10-tetraazacyclododecane) employed in makingcertain of the macrocyclic aminophosphonic acids is a commerciallyavailable material. The preparation of the macrocyclic aminophosphonicligand of this invention can also be found U.S. Pat. No. 5,059,412entitled “Macrocyclic Aminophosphonic Acid Treatment of CalcificTumors”; by Simon et al., the disclosure of which is hereby incorporatedby reference.

The preparation of these ligands have been described in several U.S.patents such as, U.S. Pat. No. 4,973,333, U.S. Pat. No. 4,882,142, U.S.Pat. No. 4,853,209, U.S. Pat. No. 4,898,724, U.S. Pat. No. 4,897,254,U.S. Pat. No. 5,587,451, U.S. Pat. No. 5,714,604, U.S. Pat. No.5,064,633, U.S. Pat. No. 5,587,451, U.S. Pat. No. 5,066,478, U.S. Pat.No. 5,300,279, U.S. Pat. No. 5,059,412, and U.S. Pat. No. 5,064,633. Thepreferred ligands useful for practicing the present invention areselected from the group consisting ofethylenediaminetetramethylenephosphonic acid (EDTMP),diethylenetriaminepentamethylenephosphonic acid (DTPMP),hydroxyethylethylenediaminetrimethylenephosphonic acid (HEEDTMP),nitrilotrimethylenephosphonic acid (NTMP),1,4,7,10-tetraazacyclododecanetetramethylenephosphonic acid (DOTMP),tris(2-aminoethyl)aminehexamethylenephosphonic acid (TTHMP), methylenediphosphonate, hydroxymethylene diphosphonate, hydroxyethylidenediphosphonate (HEDP); and ethane-1-hydroxy-1,1-diphosphonic acid.Preferred ligands are macrocyclic aminophosphonic acid ligands of which1,4,7,10-tetraazacyclododecanetetramethylenephosphonic acid (DOTMP) isan example. The present invention includes the use of the bone marrowsuppressing method and composition in combination with other drugsand/or radiation sources.

Radionuclide Complexes

Radionuclide complexes suitable for use in the present invention musthave particular properties to be suitable as therapeutic agents. Theradionuclide complex must be taken up preferentially by bone so that itis possible to deliver a bone marrow suppressing dose of radiation tothe bone marrow with minimal exposure to other tissues such as lung,liver, bladder or kidneys. The radionuclide complex also should berapidly taken up by bone and rapidly cleared from the blood, therebyfurther reducing exposure to non-target tissues.

The radionuclide and ligand can be combined under any conditions thatallow the two to form a complex. Generally, mixing in water at acontrolled pH (the choice of pH is dependent upon the choice of ligandand radionuclide) is all that is required. The complex is formed bychelation of the radionuclide by an electron donor group or groups thatresults in a relatively stable radionuclide complex, e.g., stable to thedisassociation of the radionuclide from the ligand. For example,¹⁶⁶Ho-DOTMP is formed by adding a ¹⁶⁶Ho salt, such as the chloride ornitrate in aqueous HCl (0.1-1N), to a sterile, evacuated vial containingat least 3 equivalents of DOTMP in aqueous base (KOH, NaOH and thelike). After stirring at a pH=10.5, the pH is then adjusted to 7-8 byadding phosphate buffer and a stabilizing agent such as ascorbic acid.Complexation of >99% is achieved.

For the purpose of the present invention, bone marrow suppressingradionuclide compositions described herein and physiologicallyacceptable salts thereof are considered equivalent. Physiologicallyacceptable salts refer to the acid addition salts of those bases whichwill form a salt with at least one acid group of the ligand or ligandsemployed and which will not cause significant adverse physiologicaleffects when administered as described herein. Suitable bases include,for example, the alkali metal and alkaline earth metal hydroxides,carbonates, and bicarbonates such as, for example, sodium hydroxide,potassium hydroxide, calcium hydroxide, potassium carbonate, sodiumbicarbonate, magnesium carbonate and the like, amine hydroxides,carbonates, and bicarbonates such as, for example, ammonium hydroxide,ammonium carbonate, and the like, or primary secondary and tertiaryamine hydroxides, carbonates, and bicarbonates such as, for example,trimethyl ammonium carbonate and the like. Physiologically acceptablesalts can be prepared by treating the macrocyclic aminophosphonic acidwith an appropriate base.

The macrocyclic aminophosphonic acid complexes when formed atapproximately a ligand to metal molar ratio of 1:1 to 20:1 givebiodistributions that are consistent with those exhibited by knownagents that are bone-specific. The preferred bone marrow suppressingradionuclide composition utilizes ¹⁶⁶Ho with DOTMP. Preferably, molarratios of DOTMP to ¹⁶⁶Ho are above 3, e.g., 3.5-5:1. The most preferredratio is about 3.5:1. Such ratio provides adequate complexation of theradionuclide while compensating for radiolysis of the ligand. Lowerratios of DOTMP to ¹⁶⁶Ho are unstable in vivo and not therapeuticallyeffective. By contrast, other acyclic aminophosphonic acid complexes canresult in substantial localization of radioactivity in soft tissue(e.g., liver) if large excess amounts of ligand are not used. Largeexcesses of ligand are undesirable since uncomplexed ligand may be toxicto the patient or may result in cardiac arrest or hypocalcemicconvulsions. In addition, the macrocyclic aminophosphonic acid ligandsare useful when large amounts of metal are required (i.e. for metalsthat have a low specific activity). In this case, the macrocyclicaminophosphonic acid ligands have the ability to deposit more tolerabledoses of radioactivity in the bone than is possible when usingnon-cyclic aminophosphonic acid ligands.

Stabilizing Agents

A pharmaceutically acceptable means of protecting the radionuclidecomplex from radiolytic decay of the chelator is highly preferred.Preferred radioprotectants of the present invention are radio-stableanti-oxidants, compounds that either reduce the number or the activityof oxidizing radicals. Exemplary radioprotectants that can be employedin the practice of the present invention are ascorbic acid, gentisicacid, nicotinic acid, ascorbyl palmitate, HOP(:O)H₂, monthioglycerol,sodium formaldehyde sulfoxylate, Na₂S₂O₅, Na₂S₂O₃SO₂, or a reducingagent combined with BHA, BHT, pyrogallate or tocopherol and the like.Ascorbic acid is the preferred radioprotectant for use in the practiceof the present invention, and can be used at about 35-75 mg/mL of liquidcomposition. This concentration of ascorbate can provide a solution of¹⁶⁶Ho-DOTMP that is stable, e.g., therapeutically useful, for at last 72hours, at ambient conditions, e.g., unfrozen.

The formulations of the present invention are in the solid or preferablyliquid form containing the active radionuclide complexed with theligand. These formulations can be in kit form such that the chelator andradionuclide are mixed at the appropriate time prior to use in asuitable liquid carrier with the radioprotectant. Whether premixed or asa kit, the formulations usually require a pharmaceutically acceptablecarrier, such as water.

Pharmaceutical Dosage Forms

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile solutions, dispersions, emulsions, or microemulsions,comprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in protective matrices such asnanoparticles or microparticles. In all cases, the ultimate dosage formmust be sterile, fluid and stable under the conditions of manufactureand storage. The liquid carrier or vehicle can be a solvent or liquiddispersion medium comprising, for example, water, ethanol, a polyol (forexample, glycerol, propylene glycol, liquid polyethylene glycols, andthe like), DMSO, and suitable mixtures thereof. In many cases, it willbe preferable to include isotonic agents, for example, sugars, buffersor sodium chloride.

Injectable suspensions as compositions of the present invention requirea liquid suspending medium, with or without adjuvants, as a carrier. Thesuspending medium can be, for example, aqueous polyvinylpyrrolidone,inert oils such as vegetable oils or highly refined mineral oils, oraqueous carboxymethylcellulose solutions. If necessary to keep thecomplex in suspension, suitable physiologically acceptable adjuvants canbe chosen from among thickeners such as, for example,carboxymethylcellulose, polyvinylpyrrolidone, gelatin, and thealginates. Many surfactants are also useful as suspending agents, forexample, lecithin, alkylphenol, polyethylene oxide adducts,naphthalenesulfonates, alkylbenzenesulfonates, and the polyoxyethylenesorbitan esters. Many substances that effect the hydrophobicity,density, and surface tension of the liquid suspension medium can assistin making injectable suspensions in individual cases. For example,silicone antifoams, sorbitol, and sugars are all useful suspendingagents.

Dosages of the Radionuclide Complexes

The “bone-marrow suppressing amount” or other effective therapeuticamount of radionuclide composition administered to achieve bone marrowsuppression will vary according to factors such as the age, weight andhealth of the patient, the disease state being treated, the treatmentregimen selected, as well as the nature of the particular radionuclidecomposition to be administered. For example, less activity will beneeded for radionuclides with longer half lives. The energy of theemissions will also be a factor in determining the amount of activitynecessary. Preferably, a total dose of about 20-60 Gy, most preferablyabout 30-60 Gy, e.g., 40-50 Gy of radiation will be delivered to bonemarrow via bone localization.

The radiation exposure is reported using the Grey scale (Gy) and istypically determined using a diagnostic dose of about 1200-2000 MBq(about 30 mCi to about 50 mCi) of the radionuclide/ligand. Determinationof the doses of radiation delivered by the present complexes can bedetermined in accord with the methodologies of M. Bardies et al.,Physics in Medicine and Biology, 41, 1941 (1996); J. Bayouth, RadiationPhysics, University of Texas—Houston Graduate School of BiomedicalScience: 111 (1993); A. H. Beddoe et al., Physics in Medicine & Biology,21, 589 (1976); R. Bigler et al., Health Physics, 31, 213 (1976); R.Champlin et al., Semin. Hematol, 24, 55 (1987); R. E. Champlin et al.,Cancer Treatment Reports, 68, 145 (1984); K. Eckerman et al., Journal ofNuclear Medicine, 35, 112P (1994); T. E. Hiu et al., Proceedings ofInternational Conference on Radiation Dosimetry and Safety, Taipei,Taiwan, American Nuclear Society (1987); I.C.R.P Report of the taskgroup on reference man: anatomical, physiological and metaboliccharacteristics. New York, Pergamon Press (1973); R. L. Loevinger etal., MIRD Primer for Absorbed Dose Calculations, New York, Society ofNuclear Medicine (1991); F. W. Spiers et al., British Journal ofRadiology, 54, 500 (1981); S. R. Thomas et al., J. Nucl. Med., 35, 73(1994)],” Journal of Nuclear Medicine, 33, 783 (1992).

Table 1 indicates the dosage levels achieved at various levels ofskeletal uptake of the radionuclide. TABLE 1 DOSAGE LEVELS AT PERCENTSKELETAL UPTAKE Dose Level vs. Dose Required in MBq/kg¹ Dose Level 15%Uptake 30% Uptake 45% Uptake 20 Gy 1110 518 370 30 Gy 1665 777 555 40 Gy2220 1036 740 50 Gy 2775 1295 925¹Average skeletal uptake in patients is about 30%.

The radiation amounts herein are reported in megabecquerels (MBq), Gy,or in mCi. The conversion between mCi and MBq for an average patient isillustrated below:22.0 mCi/kg X 70 kg X 37 MBq/mCi=56,980 MBq (or 814 MBq/kg).wherein 70 kg is used as an average patient weight. Herein both termshave been used. A becquerel is 1 disintegration per minute (dpm).

The mean absorbed dose to a target tissue from activity within a sourceorgan for ¹⁶⁶Ho and other radionuclide can be calculated using thegeneral method defined by the Medical Internal Radiation Dose (MIRD)committee of the Society of Nuclear Medicine. The MIRD organ scaleformalism simplifies this relationship for a radiation source intrabecular bone irradiating bone marrow to: $\begin{matrix}{\overset{\_}{D} = {\overset{\sim}{A} \cdot {S\left( {BM}\leftarrow{TB} \right)}}} & {{Equation}\quad 1} \\{{{Where}:{S\left( {BM}\leftarrow{TB} \right)}} = {\sum\limits_{i}{{\Phi_{i}\left( {BM}\leftarrow{TB} \right)} \cdot \Delta_{i}}}} & {{Equation}\quad 2} \\{{{and}:{\Phi_{i}\left( {BM}\leftarrow{TB} \right)}} = \frac{\phi\left( {BM}\leftarrow{TB} \right)}{m_{BM}}} & {{Equation}\quad 3}\end{matrix}$Where:

-   -   {overscore (D)}=Mean absorbed dose to the target organ of mass m    -   Ã=Total number of radioactive transitions within the source        organ n_(i)·E_(i)=Δ_(i)=Amount of energy released per transition        per specific radiation φ_(i)(T←S)=Fraction of energy emitted        from source organ absorbed in target organ for the specific        radiation    -   m_(BM)=Mass of bone marrow    -   S(BM←TB)=S-value for trabecular bone surfaces irradiating        adjacent bone marrow.

The distribution of radioactive material are calculated from whole bodygamma camera emission images of ¹⁶⁶Ho-DOTMP, and the rate of clearancefrom the skeleton will be measured from whole body retention data from¹⁶⁶Ho-DOTMP. S-values were generated for the Standard Man Phantom asdefined by ICRP 23 using the revised bone and bone marrow model includedin the software package MIRDOSE 3.1, and all values of radiation dose tobone marrow are calculated using this package. The calculation ofresidence time of ¹⁶⁶Ho, is important, since from the residence time,the cumulated activity or Ã can be determined. Ã and S-values are thenused to calculate absorbed dose (cGy/mCi) to the active bone marrow(Equation 1).

Independent methods can be used to estimate the total amount of skeletalradioactivity and its rate of disappearance from the skeleton, includinggamma camera serial whole body imaging, external probe whole bodyretention measurements, and gamma counter counting of cumulative urineinterval samples acquired 24 or 48 hrs. after injection.

More specifically, the following data for the calculations describedherein should be obtained:

-   -   1. Whole body serial quantitative ¹⁶⁶Ho-DOTMP diagnostic test        dose scans over 24 hr to assess DOTMP pharmacokinetics and        bio-distribution;    -   2. Cumulative urine collection and background subtracted whole        body probe measurements over 48 hr; and    -   3. Serial whole blood sampling over 24 hr (10, 30 min, 1, 2, 4,        and 24 hr).

Absorbed dose to the bone marrow is estimated by extrapolating latewhole body clearance curve time points (>12 hr) back to time=0 to makean estimate of initial bone uptake and rate of clearance for furthercalculation of bone residence time and cumulated activity. If activitywithin the total body at greater than 12 hr is due to skeletal activityonly, then the later time points for the whole body retention curvecalculated from the serial probe and cumulative urine measurements canbe used to extrapolate the initial skeletal uptake (PID(t=0)). The rateof skeletal clearance based (T_(1/2Effective)) upon a mono-exponentialfunction from which skeletal residence time can be determined. Totalskeletal residence time (RT) is calculated using equation 4.Skeletal RT=1.44·PID(t=0)·T _(1/2) ^(Effective)  Equation 4The total residence time is assumed to be distributed equally betweencortical and trabecular bone surfaces (ICRP 1973). Once the residencetimes are calculated, the therapy dose of ¹⁶⁶Ho are determined usingEquation 5: $\begin{matrix}{\begin{matrix}{{\,^{166}{Ho}} - {DOTMP}} \\{{therapy}\quad{{dose}({mCi})}}\end{matrix} = \frac{{projected}\quad{therapy}\quad{{dose}({cGy})}}{{marrow}\quad{{dose}\left( {{cGy}/{mCi}} \right)}}} & {{Equation}\quad 5}\end{matrix}$

The above calculations can be combined with methods for soft tissuedosimetry, to minimize the does to non-target tissues such as theurinary bladder wall.

The results of patients treated with 20 30 and 40 Gy with 140 mg/mL and200 mg/m² are given in Example 11.

As discussed above, and exemplified below, the administered dose ofradiation can be calculated by pre-administration of a diagnostic doseof a radionuclide complex. Depending on the percent bone uptake of agiven radionuclide complex by a given subject, which is generally in therange of about 15 to about 45%, the range of activity per administereddose can generally be from about 250 to about 3000 megabecquerels perkilogram of body weight of said mammal. If uptake is low, or if a veryhigh dose is desired, a dose of from about 750 to about 2500megabecquerels per kilogram of body weight of said mammal, or from about1000 to about 2000 megabecquerels per kilogram of body weight of saidmammal may be prefered The effective amount used to obtain bone marrowsuppression will typically be administered, generally by administrationinto the bloodstream, in a single or multi-dose infusion.

Bone Marrow Transplantation and Restoration

The general techniques of autologous or allogeneic bone marrowtransplantation or “rescue” are well known in the art, see for example,F. R. Appelbaum et al., “The Role of Marrow Transplantation in theTreatment of Leukemia”, (pp. 229-262), C. D. Bloomfield (ed.), Chronicand Acute Leukemias in Adults, Martinus Nijhoff Publishers, Boston(1985); E. D. Thomas, “Clinical Trials with Bone MarrowTransplantation”, (pp. 239-253), Clinical Trials in Cancer Medicine,Academic Press, Inc. (1985); E. D. Thomas, Journal of Clinical Oncology,1, 517 (1983); E. D. Thomas et al., Annals New York Academy of Sciences,445, 417 (1985).

Under general or spinal anesthesia and using standard marrow aspirationneedles, multiple aspirates are performed from the anterior andposterior iliac crests and, occasionally, the sternum of the donor. Themarrow is placed in heparinized tissue culture media and then, usingmetal screens, filtered to remove bony spicules and fat globules and tocreate a monocellular suspension. At the time of desired administrationof the bone marrow, the marrow is infused intravenously, following whichthe marrow stem cells migrate to the marrow space, proliferate, andeventually restore normal hematopoiesis and immune function. It ispreferable to give as many bone marrow cells as possible to enhance theprospects of marrow engraftment. Following allogeneic transplant thepatient usually receives some form of immunosuppression, such as byadministration of methotrexate or cyclosporine, in an attempt to preventor at least modify graft-versus-host disease.

A more preferred method for retrieving bone marrow stem cells involvesharvesting these cells from the peripheral blood. The purity of stemcells is enhanced by techniques such as negative selection withantibodies specific for hematopoietic cell markers. In order to increasethe concentration of stem cells in the blood, patients are pretreatedwith chemotherapy, or pretreated with a colony stimulating factor suchas G-CSF, GM-CSF, or SC-CSF. These cytokines are also used after TBI andmarrow or stem cell transplant to enhance engraftment.

The use of high dose chemotherapy followed by stem cell support hasbecome one of the most attractive therapeutic approaches in multiplemyeloma since, in relation to conventional chemotherapy, it increasesthe number of complete remissions (CR), duration of event free survival(EFS) and probably, overall survival (OS). In this setting of high dosechemotherapy, the use of ¹⁶⁶Ho-DOTMP to suppress (ablate) the marrow inorder to eradicate the malignant cells more effectively, requires stemcell support. With total marrow ablation using ¹⁶⁶Ho-DOTMP a stem cellrescue is required using autologous stem cells collected prior totherapy.

Preferably, autologous stem cells or bone marrow cells are purged ofcancerous plasma cells or tumor cells by methods known to the art, suchas binding the plasma cells with antibody-toxin conjugates or CD34selection for stem cell enrichment. The ability to give back thepatients stem cells post ablative therapy helps to regenerate the hosthematopoiesis and thus protect the patient from potentiallylife-threatening complications. In the case of multiple myeloma patientstreated with the present method, e.g., high dose melphalan and 40-50 Gyof radiation to bone marrow from ¹⁶⁶Ho-DOTMP, the high efficiency ofbone marrow suppression effectively increases the negative effect ofresidual cancer cells in autologous marrow. Therefore, purgingautologous cells can improve the outcome for such patients.

Treatment of Cancer

A. Chemotherapeutic Agents

In the treatment of a patient having a cancer such as leukemia ormultiple myeloma, the use of the radionuclide compositions describedherein can reduce or eliminate the neoplastic cell population in thebone marrow. The aminophosphonate ligands also lead to enhanced uptakeof the radionuclide by neoplastic bone lesions, which represent areas ofactive bone matrix turnover. However, it will usually be necessary toadminister one or more chemotherapeutic agents, to destroy theundesirable cells in locations other than the bone marrow or insanctuaries within the bone marrow, or to add to the effects of theradiation. The efficacy of cancer elimination can be enhanced by the useof protein synthesis inhibitors, in order to inhibit repair of damagedDNA in the cancer cells.

Chemotherapeutic antineoplastic (“anti-cancer”) agents that are usefulin practicing the present invention include but are not limited todoxorubicin, fludarabine, ifosfamide, thiotepa, melphalan(L-phenylalanine, 4-[bis(2-chloroethyl)amino]-), methotrexate,mitoxantrone, estramustine, bleomycin, vinblastine, taxanes,thalidomide, etoposide, tamoxifen (anti-estrogens) (10-20 mg 2×daily forbreast cancer), paclitaxel, vincristine, dexamethasone, busulfan,cyclophosphamide, bischloroethyl nitrosourea, cytosine arabinoside,6-thioguanine, organoplatinum-based agents and analogs thereof.Preferred chemotherapeutic agents that are useful in practicing thepresent invention, particularly with respect to metastatic breast cancerare doxorubicin, thiotepa, melphalan, methotrexate, bleomycin,vinblastine, taxol, taxanes, tamoxifen, busulfan and analogs thereof.Preferred chemotherapeutic agents, particularly for the treatment ofmetastatic prostate cancer include mitoxantrone, estramustine,adriamycin and taxanes. Hormone (e.g., anti-androgren) treatment canalso be employed to inhibit the spread of prostrate cancer, as can useof non-steroidal aniinflammatory agenst such as etodolac.

Preferred chemotherapeutic agents for treatment of multiple myelomainclude melphalan, thalidomide, vincristine, doxorubicin, dexamethosoneand doxorubicin.

The present method is particularly advantageous in that it can be usedwith chemotherapeutic agents, such as alkylating agents, that alsosuppress bone marrow. For example, melphalan analogs are disclosed inU.S. Pat. Nos. 3,032,584 and 3,032,585 (see Merck Index (11th ed.) atpage 914). Conventional dosages and dosage forms of melphalan aredisclosed at page 1154 of Remington's Pharmaceutical Sciences, Mack Pub.Co. (18th ed. 1990).

The term “chemotherapeutic agent” also includes anti-cancer agents, suchas toxins, that are targeted to cancer cells by antibodies againstcancer cell antigens. Such immunoconjugates are described in publishedPCT applications WO/97/00476 and WO/95/10940. The term chemotherapeuticagent also includes monoclonal antibody based therapies such asherceptin and rituxan (rituximab).

In conjunction with the present method chemotherapy can be given instandard doses; preferably, chemotherapy is given at the upper limit ofthe conventional ranges or at higher than standard doses, depending onthe tolerance of the patient. Standard doses for representativechemotherapeutic agents are shown in the following Table A. TABLE AChemotherapeutic Agent Dose* Doxorubicin 60-120 mg/m²/day Fludarabine30-350 mg/m²/day Ifosfamide 5-10 g/m² (single dose) Thiotepa 1.5-500mg/m²/day Methotrexate 12-500 mg/m² i.v. Mitoxantrone 10-30 mgEstramustine 50-1120 mg/day Bleomycin 10-30 U/m² Vinblastine 5-10 mg/m²Docetaxol 50-200 mg/m² i.v. Thalidomide 100-1000 mg/day Paclitaxel135-300 mg/m² Etoposide 100-5400 mg/m²/day Tamoxifen 20-60 mg/dayVinorelbine 20-100 mg/m²/day Vincristine 1-2 mg/m²/day Dexamethazone10-60 mg/day Busulfan 12-16 mg/kg/day Cyclophosphamide 750-6000 mg/m²Carmustine 250-600 mg/m² i.v. Cytosine arabinoside 50-200 mg/m²/dayCarboplatin 100-500 mg/m²/day AUC 4-12 day*Ranges from low dose given per day over multiple days to single highdaily dose.

B. Adjunct Agents

The mammals (patients) can also be pre-treated with agents such asbisphosphonates, to counteract the hypercalcemia associated with certaintumors, such as lung cancers, multiple myeloma, renal cell carcinoma,bronchogenic carcinoma, breast cancer, lymphoma, and cancers of the headand neck. Pamidronate, clodronate, zaledronate, etidronate, tiludronateand alendronate are preferred agents for treatment of this condition. Itwill be appreciated that the agents should be selected and used so asnot to compete with the therapeutic agent for bone uptake.

The mammals (patients) can be hydrated and premedicated with antiemeticsto decrease nausea and vomiting that may be associated with suppressionof bone marrow when practicing the present invention. The preferredantiemetics are those that reduce the irritation of the chemoreceptortrigger zone such as Zofran®. Common regimens that are useful inpracticing the present invention include serotonin 5-HT³ antagonistssuch as, for example, ondansteron, granisetron, and the like; dopamineantagonists such as, for example, prochlorperazine, promethazine,droperidol, metoclopramide, and the like; antihistamines andanticholinergics such as, for example, diphenylhydramine, scopolamine,dimethylhydrinate, meclizine, and the like; corticosteroids such as, forexample, dexamethasone and the like; and sedatives such as, for example,diazepam, lorazepam, and the like.

C. Cancers Subject to Treatment

A wide variety of leukemias and tumors can be treated with the presentcomplexes including bone-forming or calcific tumors, and fibro-osseoustumors, leukemias such as chronic lymphocytic leukemia and myeloidleukemia, and metastatic tumors to the skeleton. Skeletal system tumorsinclude, but are not limited to, sarcomas such as Ewing's sarcoma,osteochondroma, sarcoma of the periosteum, osteosarcoma, osteoma,osteoblastoma, chondrosarcoma, and giant cell tumor of the bone. Othertumors which can be treated include chordoma, adamanthoma,hemangioendothelioma, hemangiopericytoma, myelomas, such as multiplemyeloma, non-Hodgkin's lymphoma, Hodgkin's disease, breast cancer,prostate cancer, lung cancer, head and neck cancer, ovarian cancer,bladder cancer, liver cancer, pancreatic cancer, renal cell carcinoma,myelodysplastic syndrome, germ cell tumor, and neuroblastoma,particularly those cancers that have metastasized to the bone, attach tothe bone, or that are associated with the skeletal system. The presentmethod is particularly well-suited for the treatment of various formsand stages of multiple myeloma. Such forms and stages of multiplemyeloma are discussed in R. Bataille et al., cited above.Myeloproliferative disorders that are not necessarily classified ascancers, including polycythemia vera, macroglobulinemia, megakaryocyticmyelosis or malignant histiocytosis, can also be treated with thepresent complexes.

D. Adjunct Radiation Therapy

By careful aiming and regulation of dose, high-energy radiation can beused to destroy cancer cells in combination with the presentradionuclide therapy. Radiation therapy (also referred to asradiotherapy, x-ray therapy, cobalt treatment, or irradiation) ispresently either part of the treatment or the only treatment for abouthalf of all cancer patients. This form of treatment is effective onlyfor those cancer cells within the area receiving the radiation (thefield), which can encompass the entirety of the subject's body (totalbody irradiation or TBI) or can be localized, as in the exposure of aspecific tumor site.

Radiation may be used before surgery to shrink a cancerous tumor, aftersurgery to stop growth of any remaining cancer cells, or alone or withanticancer drugs to destroy a malignant tumor. It is particularlyeffective when used to treat certain types of localized cancers such asmalignant tumors of the lymph nodes or vocal cords.

Radiation usually is not per se curative if the cancer cells have spreadthroughout the body or outside the area of radiation. It can be usedeven if a cure is not probable because it can shrink tumors, whichdecreases the pressure and pain they cause, or it can stop tumorbleeding.

Generally, radiation produces less physical disfigurement than radicalsurgery, but it may produce severe side effects. These side effects arerelated to the damage x-rays do to normal tissue such as blood or bonemarrow. Side effects include irritated skin, swallowing difficulties,dry mouth, nausea, diarrhea, hair loss, and a loss of energy. Howserious and extensive these side effects become depend on where and howmuch radiation is used.

Use of the present radionuclide complexes can reduce or eliminate theneed for total or targeted external radiation therapy, or can enhancethe total efficacy of a therapeutic regimen that normally employs TBI.Doses of TBI useful in the present method can deliver total irradiationof from about 750-1350 cGy, e.g., about 800-1000 to 1200 cGy. The totalirradiation may be given in multiple fractions, i.e., 1-10 fractions; orin a single dose.

Treatment of Autoimmune Diseases and Immunosuppression

The methods and compositions of the invention are also useful to treatimmunologic disorders such as autoimmune diseases by immune suppressiondue to temporary partial bone marrow suppression or by marrow purging,in combination with marrow transplantation. However, those skilled inthe art would recognize that the methods and compositions of theinvention can also be used for general immunosuppression in combinationwith other immunosuppressive therapies. Currently, autoimmune diseasesare treated by a variety of nonspecific immunosuppressive drugs andsteroids. One group of anti-inflammatory agents used in the treatment ofautoimmune diseases is corticosteroids. Corticosteroids are syntheticversions of the body's hormone cortisone, which is produced in smallamounts by the adrenal gland. Synthetically produced corticosteroidsreduce inflammation and suppress the immune system. The most commonlyprescribed corticosteroids for use in treating autoimmune disorders areprednisone and dexamethasone.

Autoimmune disorders are sometimes treated with immunosuppressant drugssuch as cytotoxic agents (e.g. methotrexate, azathioprine andcyclophosphamide). In addition, anti-malarials including chloroquine andhydroxychloroquine can be used to suppress inflammation and the immunesystem in the treatment of autoimmune disorders. Autoimmune diseases canalso be treated with nonsteroidal anti-inflammatory medications, such asaspirin, ibuprofen, naproxen, indomethacin, sulindac, etodolac andtolmetin. Gold salts have been used to treat autoimmune arthritis forover a half a century, while recent advances in research have yieldednew autoimmune arthritis therapies, such as COX-2 inhibitors. COX-2inhibitors (or super-aspirin) work to inhibit inflammation and painwithout producing significant side effects. In addition, another classof agents that target aberrant cytokine production, such as anti-TNF(tumor necrosis factor) drugs, can also be used for the treatment forseveral types of autoimmune diseases including rheumatoid arthritis,lupus, myositis, and scleroderma.

Furthermore, the methods and compositions of the invention could also beused alone or in combination with drugs that act more specifically onthe immune system, for example, by blocking a particularhypersensitivity reaction. In addition, the complexes could be used incombination with intravenous immunoglobulin therapy or otherantibody-based therapies, a treatment, used for various immunologicaldiseases to reduce circulating immune complexes, or specific T cellpopulations. For example, the present methods and complexes can be usedas immunosuppressive agents to inhibit host rejection of transplantedcells, tissue or organs.

In order to increase the chance of the patient's recovery, it can bebeneficial to employ hematopoietic cell growth factors, such asgranulocyte macrophage colony stimulating factor (GM-CSF), orgranulocyte colony stimulating factor (G-CSF), and IL-11 forthrombopoiesis to stimulate or enhance the regeneration and restorationof the bone marrow. It can also be beneficial to employ stem cell growthfactor, G-CSF and/or GM-CSF prior to therapy to trigger release of stemcells into the blood where they can be collected.

Infections and Infectious Diseases

The methods and compositions of the invention are also effective totreat bacterial infections, fungal infections, parasitic infections, andinfectious diseases that localize to or around bone such astuberculosis, syphilis, bacterial osteomyelitis, fungal osteomyelitisfor example blastomycosis and cryptococcosis, and the like. Anti-fungalagents, and anti-bacterial agents often have poor penetration into thebone and sites enclosed by bone such as the bone marrow. In situationsin which the patient is suffering from an infectious disease that haslocalized to the bone, the patient may be able to achieve a cure by thedelivery of high doses of radiation to the bone.

Examples of agents useful in combination with targeted radiation inpracticing the present invention include, but are not limited toantibiotic agents, e.g., antibacterial urinary tract agents;anti-infective agents, anti-parasitic agents and anti-fungal agents,including those disclosed in The Physician's Desk Reference, 50thEdition, 1996.

Useful antibiotic agents include systemic antibiotics, such asaminoglycosides, cephalosporins (e.g., first, second, and thirdgeneration), macrolides (e.g., erythromycins), monobactams, penicillins,quinolones, sulfonamides, and tetracyclines, including those disclosedin The Physician's Desk Reference, 50th Edition, 1996.

In addition, antibacterial agents include 2-isocephem and oxacephemderivatives disclosed in U.S. Pat. No. 5,919,925; pyridonecarboxylicacid derivatives disclosed in U.S. Pat. No. 5,910,498; water miscibleesters of mono- and diglycerides disclosed in U.S. Pat. No. 5,908,862;benzamide derivatives disclosed in U.S. Pat. No. 5,891,890;3-ammoniopropenyl cephalosporin compounds disclosed in U.S. Pat. No.5,872,249; 6-O-substituted ketolides disclosed in U.S. Pat. No.5,866,549; benzopyran phenol derivatives disclosed in U.S. Pat. No.5,861,430; pyridine derivatives disclosed in U.S. Pat. No. 5,859,032;2-aminothiazole derivatives disclosed in U.S. Pat. No. 5,856,347; penemester derivatives disclosed in U.S. Pat. No. 5,830,889;lipodepsipeptides disclosed in U.S. Pat. No. 5,830,855; dibenzimidazolederivatives disclosed in U.S. Pat. No. 5,824,698; alkylenediaminederivatives disclosed in U.S. Pat. No. 5,814,634; organicsolvent-soluble mucopolysaccharides disclosed in U.S. Pat. No.5,783,570; arylhydrazone derivatives disclosed in U.S. Pat. No.5,760,063; carbapenem compounds disclosed in U.S. Pat. No. 5,756,725;N-acylpiperazine derivatives disclosed in U.S. Pat. No. 5,756,505;peptides disclosed in U.S. Pat. No. 5,714,467; oxathiazines and theiroxides disclosed in U.S. Pat. No. 5,712,275; 5-amidomethyl alphabeta-saturated and -unsaturated 3-aryl butyolactone compounds disclosedin U.S. Pat. No. 5,708,169; halogenated benzene derivatives disclosed inU.S. Pat. No. 5,919,438; sulfur-containing heterocyclic compoundsdisclosed in U.S. Pat. No. 5,888,526; and oral antibacterial agentsdisclosed in U.S. Pat. No. 5,707,610.

Anti-parasitic agents include agents capable of killing arthropods(e.g., lice and scabies); helminths (e.g., ascaris, enterobius,hookworm, stronglyoids, trematodes, and trichuris); and protozoa (e.g.,amebas, malaria, toxoplasma, and trichomonas), including those disclosedin The Physician's Desk Reference, 50th Edition, 1996.

The methods and compositions of the invention are also effective totreat fungal infections that localize to or around bone such as fungalosteomyelitis and the like. The methods and compositions can also beused in conjunction with antifungal agents known to be useful in thetreatment of fungal infections. Antifungal agents include dermatologicalfungicides, topical fungicides, systemic fungicides, and vaginalfungicides, including those disclosed in The Physician's Desk Reference,50th Edition, 1996.

In addition, antifungal agents include terpenes, sesquiterpenesditerpenes, and triterpenes disclosed in U.S. Pat. No. 5,917,084;sulfur-containing heterocyclic compounds disclosed in U.S. Pat. No.5,888,526; carbozamides disclosed in U.S. Pat. No. 5,888,941;phyllosilicates disclosed in U.S. Pat. No. 5,876,738; corynrcandinderivatives disclosed in U.S. Pat. No. 5,863,773; sordaridin derivativesdisclosed in U.S. Pat. No. 5,854,280; cyclohexapeptides disclosed inU.S. Pat. No. 5,854,213; terpene compounds disclosed in U.S. Pat. No.5,849,956; agents derived from aspergillus fumigatus disclosed in U.S.Pat. No. 5,873,726; inula extracts disclosed in U.S. Pat. No. 5,837,253;lipodepsipeptides disclosed in U.S. Pat. No. 5,830,855; polypeptidesdisclosed in U.S. Pat. No. 5,824,874; pyrimidone derivatives disclosedin U.S. Pat. No. 5,807,854; agents from sporomiella minimizes disclosedin U.S. Pat. No. 5,801,172; cyclic peptides disclosed in U.S. Pat. No.5,786,325; polypeptides disclosed in U.S. Pat. No. 5,773,696; triazolesdisclosed in U.S. Pat. No. 5,773,443; fusacandins disclosed in U.S. Pat.No. 5,773,421; terbenzimidazoles disclosed in U.S. Pat. No. 5,770,617;and agents obtained from hormones disclosed in U.S. Pat. No. 5,756,472.

Pathologies Treatable by BMT or Stem Cell Replacement

The present methods can be useful to ablate bone marrow in treatmentregimens intended to correct a variety of disorders by replacing“defective” hematopoietic cells, with “normal” autologous or allogeneicbone marrow or stem cells. This can be used in the treatment of diseasesof red cells and bleeding disorders. These include hematopoietic geneticdiseases such as hemolytic anemias, i.e., sickle cell anemia orthalassemia. Other such disorders include various anemias, polycythemia,thrombocytopenia, and bleeding disorders related to defective plateletfunction or abnormalities in clotting factors.

Hematopoietic stem cell transplantation from normal donor has beenreported to be effective to treat lysosomal and peroxisomal storagediseases, such as globoid cell leukodystrophy, metachromaticleukodystrophy, adrenoleukodystrophy, mannosidosis, flucosidosis,aspartylglucosaminuria; Harder, Maroteaux-Lamy and Sly Syndromes andGaucher disease type III. W. Krivit et al., Curr. Opin. Neurol., 12, 167(1999).

Gene Therapy

The present method can also be employed as part of gene therapy thatinvolves implantation of genetically engineered stem cells, to correctgenetic defects, following bone marrow ablation. For example, asubject's own stem cells can be “normalized” by introduction of a vectorcomprising a gene that will effectively counteract the defective gene orreplace the missing one. See, D. B. Kohn, Curr. Opinion in Pediatr., 7,56 (1995).

Bone marrow suppression, followed by administration of geneticallyengineered (transformed) stem cells, can be used, for example, in thetreatment of cancer in a human by inserting exogenous genes into humanprimary cells, such as, for example, stem cells, which specifically“target” mature blood cells to a tumor. Preferably, the stem cells havebeen removed from a cancer patient and expanded in culture. Genes thatenhance the anti-tumor effects of the mature cells can also be employed.The blood cells can be expanded in number before or after insertion ofthe genes. A method for transforming blood cells is described in U.S.Pat. No. 5,286,497. Thus, the procedure is performed in such a mannerthat upon injection into the patient, the transformed blood cells willproduce an anti-cancer agent in the patient's body, preferably at thesite of the tumor itself.

The gene carried by the transformed stem cells can be any gene thatdirectly or indirectly enhances the therapeutic effects of the resultantmature blood cells such as a recombinant normal human gene. The genecarried by the stem cells can be any gene that allows the blood cells toexert a therapeutic effect that it would not ordinarily have, such as agene encoding a clotting factor useful in the treatment of hemophilia.Examples of other suitable genes include those that encode cytokinessuch as TNF, interleukins (interleukins 1-12), interferons (α, β,γ-interferons), T-cell receptor proteins and Fc receptors forantigen-binding domains of antibodies, such as immunoglobulins.

Additional examples of suitable genes include genes that modify bloodcells to “target” to a site in the body to which the blood cells wouldnot ordinarily “target,” thereby making possible the use of the bloodcell's therapeutic properties at that site. In this fashion, blood cellscan be modified, for example, by introducing a Fab portion of amonoclonal antibody into the stem cells, thereby enabling the matureblood cells to recognize a chosen antigen. Other genes useful in cancertherapy can be used to encode chemotactic factors that cause aninflammatory response at a specific site, thereby having a therapeuticeffect. Other examples of suitable genes include genes encoding solubleCD4 which is used in the treatment of AIDS and genes encodingpreselected polypeptides or protein that can act to correct orameliorate genetic disorders which result in insufficient or defectiveenzymes. Such genes include the α-antitrypsin gene, which is useful inthe treatment of emphysema caused by α-antitrypsin deficiency, atyrosine hydroxylase gene (Parkinson's disease), a glucocerebrosidasegene (Gaucher's disease), an α-galactosidase gene (Fabray's disease) anarylsulfatase A gene (metachromatic leukodystrophies), an insulin genefor use in diabetes, or genes encoding other polypeptides or proteins.

The gene therapy of the present invention is also useful in thetreatment of a variety of diseases including but not limited toadenosine deaminase deficiency, sickle cell anemia, thalassemia,hemophilia, diabetes, α-antitrypsin deficiency, brain disorders such asAlzheimer's disease, and other illnesses such as growth disorders andheart diseases, for example, those caused by alterations in the waycholesterol is metabolized and defects of the immune system.

One of skill in the art would recognize that the conditions discussedherein above can have multiple causes and can overlap in naming andcategorization.

The following examples are included to aid in the understanding of theinvention but are not to be construed as limiting the invention.

EXAMPLE 1

¹⁶⁶Ho-DOTMP Preparation

Ho-165-nitrate targets are prepared from dissolution of holmium oxide innitric acid followed by reduction to dryness. A target containing 6 mgof holmium is irradiated in a reactor for approximately 155 hours at aflux of 4.5×10¹⁴ n/cm²/s. The specific activity is typically in therange of 1.3-2 Ci/mg.

The ¹⁶⁶Ho-nitrate target is dissolved in 0.3 N HCl. In a typical 9 Cipreparation, ¹⁶⁶Ho-chloride is supplied in 10 ml of 0.3 N HCl. Six vialsof DOTMP (each vial containing 10 mg DOTMP and 28 mg NaOH) is dissolvedin 4 ml water and added to the ¹⁶⁶Ho chloride. The ligand to metal ratiois 3.5. The reaction mixture is allowed to mix for 10 minutes at a pH of10.5. This is followed by addition of 4.8 ml of 1.0 M sodium phosphatebuffer and ascorbic acid. The final concentration of ascorbic acid is 55mg/ml. Dilution with water may be performed to assure that the finalactivity concentration does not exceed 322 mCi/ml. The pH of the finalproduct is 7-8.

EXAMPLE 2

Preparation of ¹⁵³Sm-Solution

Sm-153 is produced by irradiating 99.06 percent enriched ¹⁵²Sm₂O₃ in thefirst row reflector at a neutron flux of 8×10¹³ neutron/cm²×sec, or athigh flux of 4.5×10¹⁴ n/cm²/sec, at the Missouri University ResearchReactor (MURR). Irradiations are generally carried out for 50 to 60hours, yielding a Sm-153 specific activity of 1000-1300 Ci/g.

To irradiate Sm₂O₃ for production of Sm-153, the desired amount oftarget is first weighed into a quartz vial, the vial flame sealed undervacuum and welded into an aluminum can. The can is irradiated for thedesired length of time, cooled for several hours and opened remotely ina hot cell. The quartz vial is removed and transferred to a glove box,opened into a glass vial that is then sealed. An appropriate amount of asolution of hydrochloric acid is then added to the vial via syringe inorder to dissolve the Sm₂O₃. Once the Sm₂O₃ is dissolved, the samariumsolution is diluted to the appropriate volume by addition of water. Thesolution is removed from the original dissolution vial that contains theshards of the quartz irradiation vial, and transferred via syringe to aclean glass serum vial.

EXAMPLE 3

Preparation of ¹⁵⁹Gd Solution

Gadolinium-159 is prepared by sealing gadolinium oxide (1.1 mg) into aquartz vial. The vial is welded inside an aluminum can and irradiatedfor 30 hours in a reactor at a neutron flux of 8×10¹³ neutron/cm²×sec.The contents of the quartz vial are dissolved using HCl. Water is addedto obtain a solution of Gd-159 in 0.1N HCi.

EXAMPLE 4

Preparation of ¹⁵³Sm-DOTMP

The DOTMP ligand (22 mg) was dissolved in 878 μL of distilled water and15 μL of 50% NaOH. A volume of 15 μL of this solution was transferred toa vial containing 1.5 mL of Sm solution (0.3 mM Sm in 0.1N HCl spikedwith 2 μL of Sm-153 tracer). The pH was adjusted to 7-8 using NaOH andthe amount of Sm found as a complex was greater than 99% as determinedby ion exchange chromatography. This yielded a solution containing Sm at0.3 mM with a ligand to metal molar ratio of approximately 1.5.

EXAMPLE 5

Preparation of ¹⁶⁶Ho-DOTMP

The DOTMP ligand (22 mg) was dissolved in 878 μL of distilled water and15 μL of 50% NaOH. A volume of 30 μL of this solution was transferred toa vial containing 1.5 ml of Ho solution (0.6 mM Ho in 0.1N HCl spikedwith 2 μL of ¹⁶⁶Ho tracer). The pH was adjusted to 7-8 using NaOH andthe amount of Ho found as a complex was greater than 99% as determinedby ion exchange chromatography. This yielded a solution containing 0.6mM Ho with a ligand to metal molar ratio of approximately 1.5.

EXAMPLE 6

Pharmacokinetics and Patient Specific Dosimetry of High Dose ¹⁶⁶Ho-DOTMPTherapy used for Treatment of Breast Cancer Metastatic to Bone

Eight patients with breast cancer metastatic only to bone initiallyreceived a 30 mCi dose of ¹⁶⁶Ho-DOTMP for diagnostic purposes.Pharmacokinetics were assessed via whole body counting, gamma cameraimaging, and urine and blood assays for the first 48 hours followinginjection. Patients were followed with autologous stem celltransplantation for rescue from hematologic toxicity.

The average percentage uptake in the skeleton was 28±12% (range: 15% to47%), with an effective skeletal half-life of 19.9±2.5 hours (range: 15to 23 hours). Approximately 50% of the material was present in the urineat 6 hours post injection. Whole blood clearance was rapid and biphasic:early T_(1/2):0.05±0.04 hours: late T_(1/2):11±4 hours with, on average,a small percentage of the injected dose remaining at 24 hours postinjection.

Therapy doses were calculated based upon prescribed dose to the red bonemarrow using the Medical Internal Radiation Dose (MIRD) technique andpercentage localization in the skeleton. Appropriate S-values wereprovided by Oak Ridge National Laboratory. The desired target dose was22 Gy to the red marrow calculated by the above technique for eachindividual. The average red marrow dose was calculated to be 1.97±0.92cGy/mCi (range: 0.98 cGy/mCi to 3.19 cGy/mCi). Three patients proceededto therapy, two were disqualified due to low uptake in the skeleton(<30%; revised qualification: 15%), and three were disqualified forother reasons unrelated to the ¹⁶⁶Ho-DOTMP treatment. Other than severehematological suppression, no toxicity was noted.

EXAMPLE 7

¹⁶⁶Ho-DOTMP-Melphalan Treatment of Multiple Myeloma (MM) Patients

Multiple myeloma patients(≦65 yrs. of age) that have responded toinitial chemotherapy or have primary refractory disease or chemotherapyresponsive relapse, but who are not in refractory relapse are treated.Patients are well hydrated with fluids during the day prior to thediagnostic dose. An initial diagnostic dose of 30 mCi of ¹⁶⁶Ho-DOTMP isadministered to confirm the selective localization to the skeleton,establish the in-vivo pharmacokinetics and provide radiation dosimetryestimates for the red marrow. Assuming >15% of the ¹⁶⁶Ho-DOTMPaccumulates in bone following the injection, the amount of ¹⁶⁶Ho-DOTMPrequired for therapy is calculated based on delivering a specifiedradiation absorbed dose to the marrow. Patients receive the therapeuticdose by intravenous injection over 5-10 minutes, given over 1-3 days ≧48hrs after the dosometry (test) dose.

The time line for conducting the investigation is as follows: Test dose¹⁶⁶Ho-DOTMP (30 mCi); ¹⁶⁶Ho scan image (0, 4-6, 20-24 hr.); Bloodsamples for dosimetry (10, 30 min, 1, 2, 6, 20-24 hr.); Urine samples(0-6,6-12, 12-24, 24-48 hr.); and External whole body probe (0, 2, 6, 24and 48 hr.).

Melphalan is administered 48 hr prior to the predicted PBSC infusionbased upon dosimetry assessment from the test dose. PBSC infusion isadministered when bone marrow dose from ¹⁶⁶Ho is ≦1 cGy/hr. Patientswere treated at 20, 30, 40 and prospectively 50 Gy, and with 140 mg/m²and 200 mg/m² melphalan. The results are shown in Table 2, hereinbelow.

The MTD was defined as the level that is associated with a true toxicityrate of 20%, where toxicity for these purposes was taken to be grade 3or greater extramedullary drug related toxicity.

All toxicities encountered during the study will be evaluated accordingto Bearman criteria (Bearman et al., J Clin Oncol, 6,1562, (1988)).Graft failure is considered a grade 3 toxicity. Graft failure is definedas failure to recover granulocytes to 0.5×10⁹/l or platelets 20×10⁹/lwithin 28 days of transplant or a fall to less than these levels for 3or more consecutive days after day 28 without other apparent cause.Hematopoietic recovery (engraftment) is defined as having a sustainedgranulocyte count of 0.5×10⁹/l for two consecutive counts posttransplant and a platelet count 20×10⁹/l for seven consecutive countspost transplant, without transfusion support. The first of two countsfor the granulocyte count and the first of seven counts for the plateletcount are considered the day of engraftment.

Patients undergo blood stem cell infusion at the time when ongoingradiation to the marrow falls to <1 rad/hr, and at least 24 hours aftermelphalan infusion. The total volume of stored cells is infused into afree flowing IV line primed with normal saline. Patients arepremedicated with acetaminophen 650 mg PO and diphenhydramine 50 mg POor IV. All patients receive conventional supportive care forautologous/syngeneic blood and marrow transplantation, (such asallopurinol, menstrual suppression, prophylactic antibiotics, empiricantibiotics, IV Ig, transfusions of blood products, hyperalimentation,and the like).

EXAMPLE 8

Single Dose ¹⁶⁶Ho-DOTMP Treatment with Melphalan

Well hydrated mammals (Humans should be instructed to take in fluids inexcess of 2000 cc during the prior 24 hours.) are administered aninitial diagnostic dose of 30 mCi to confirm the selective localizationto the skeleton, establish the in-vivo pharmacokinetics, skeletaluptake, and provide radiation dosimetry estimates for the red marrow.The actual dosage is of the ¹⁶⁶Ho required for therapy will becalculated on the basis of percent uptake in the skeleton and that valueused to deliver the specified radiation absorbed dose to the marrow.Patients will receive the therapeutic dose of 20 Gy (370-1110megabecquerels per kilogram of body weight) or 30 Gy (555-1665megabecquerels per kilogram of body weight) or 40 Gy (740-2220megabecquerels per kilogram of body weight) or 50 Gy (925-2775megabecquerels per kilogram of body weight) by intravenous injectionover 2-10 minutes given on a single day. The mammals are thenadministered melphalan, 140 mg/m², 200 mg/m² or 220 mg/m², 48 hoursprior to stem cell (PBSC) infusion which occurs about 6-8 days after¹⁶⁶Ho-DOTMP administration, when the bone marrow exposure rate dropsbelow 1 cGy/hour. Mammals are started on granulocyte-colony stimulatingfactor (G-CSF) at a dose of 5-10 mcg/kg/day, and continued until thegranulocyte count is 1×10⁹/L for 3 consecutive days. Mammals are alsoadministered prophylactic antibiotic and antifungal agents whileneutropenic.

EXAMPLE 9

Single Dose ¹⁶⁶Ho-DOTMP Treatment with Melphalan and TBI

Well hydrated mammals (Humans should be instructed to take in fluids inexcess of 2000 cc during the prior 24 hours.) are administered aninitial diagnostic dose of 30 mCi to confirm the selective localizationto the skeleton, establish the in-vivo pharmacokinetics, skeletaluptake, and provide radiation dosimetry estimates for the red marrow.The actual dosage is of the ¹⁶⁶Ho required for therapy will becalculated on the basis of percent uptake in the skeleton and that valueused to deliver the specified radiation absorbed dose to the marrow.Patients will receive the therapeutic dose of 20 Gy (370-1110megabecquerels per kilogram of body weight) or 30 Gy (555-1665megabecquerels per kilogram of body weight) or 40 Gy (740-2220megabecquerels per kilogram of body weight) or 50 Gy (925-2775megabecquerels per kilogram of body weight) by intravenous injectionover 2-10 minutes given on a single day. This is then followed by TBI(800 cGy in four fractions) on successive days. The mammals are thenadministered melphalan, 140 mg/m², 48 hours prior to peripheral bloodstem cell (PBSC) infusion which occurs about 6-8 days after ¹⁶⁶Ho-DOTMPadministration, when the bone marrow exposure rate drops below 1cGy/hour. Mammals are started on granulocyte-colony stimulating factor(G-CSF) at a dose of 5-10 mcg/kg/day, and continued until thegranulocyte count is 1×10⁹/L for 3 consecutive days. Mammals are alsoadministered prophylactic antibiotic and antifungal agents whileneutropenic.

EXAMPLE 10

¹⁶⁶Ho-DOTMP Treatment

Well hydrated mammals (Humans should be instructed to take in fluids inexcess of 2000 cc during the prior 24 hours.) are administered aninitial diagnostic dose of 30 mCi to confirm the selective localizationto the skeleton, establish the in-vivo pharmacokinetics, skeletaluptake, and provide radiation dosimetry estimates for the red marrow.The actual dosage of the ¹⁶⁶Ho required for therapy will be calculatedon the basis of percent uptake in the skeleton and that value used todeliver the specified radiation absorbed dose to the marrow. Patientswill receive the therapeutic dose of 50 Gy (2000-3000 megabecquerels perkilogram of body weight) by intravenous injection over 2-10 minutesgiven on a single day. When the bone marrow exposure rate drops below 1cGy/hour mammals are started on granulocyte-colony stimulating factor(G-CSF) at a dose of 5-10 mcg/kg/day, and continued until thegranulocyte count is 1×10⁹/L for 3 consecutive days. Mammals are alsoadministered prophylactic antibiotic and antifungal agents whileneutropenic.

EXAMPLE 11

Following the procedures described in Examples 7-9, patients, afflictedwith multiple myeloma, were treated according to the method of thepresent invention. The results are described in table 2, below. Theseresults demonstrate that the combination of 200 mg/m² melphalan andHo-DOTMP is at least as efficatious or is more efficatious than thecombination of 140 mg/m² melphalan with Ho-DOTMP, either with or withoutTBI. Additionally, the combination of 200 mg/m² melphalan and Ho-DOTMPdid not produce more grade 3 toxicity than the other therapies. TABLE 2Ho-DOTMP Dose 140 mg/m² melphalan w/o TBI 140 mg/m² melphalan w/TBI 200mg/m² melphalan w/o TBI to Marrow: 20 Gy 30 Gy 40 Gy 20 Gy 30 Gy 40 Gy20 Gy 30 Gy 40 Gy Days to ANC > 500 10 16 12 9 11 9 10 12 10.5 (8-13)(13-17) (9-13) (9-16) (9-14) (9-10) (10-10) (9-19) (9-13) Days toPlatelets > 10 10 10 12 12 13 11 10 10 20,000 (7-14) (7-44) (7-19)(9-19) (7-28) (8-21) (9-19) (6-10) (7-15) Number of Patients 0/5 0/4 0/70/8 1/8 0/20 0/4 0/7 0/14 w/ Grade 3 Toxicity Complete Response 3/4 7/178/9ANC = Absolute neutrophil countNA = Not Available

The three protocols enrolling patients using ¹⁶⁶Ho for multiple myelomahave accrued 72 patients, with 66 evaluable for safety and 40 evaluablefor response. The original protocol treating patients with melphalan(140 mg/m²) without TBI was amended to increase the dose of melphalan to200 mg/m². The increase in melphalan was to determine if ¹⁶⁶Ho-DOTMPcould be given in combination with high dose melphalan without addedunmanageable toxicity.

The melphalan (140 mg/m²)/TBI/¹⁶⁶Ho-DOTMP protocol has treated 24patients total, 17 of which were evaluable.

The melphalan (140 mg/m²)/¹⁶⁶Ho-DOTMP protocol has treated 16 patients,14 of which were evaluable.

The melphalan (200 mg/m²)/¹⁶⁶Ho-DOTMP protocol has treated 32 patients,9 of which were evaluable.

The dosage range of radiation from the complex was 460 mCi to 4.5 Ci.

To date there have been no major, unexpected extramedullary toxicitiesrelated to therapy >grade 2. One patient had graft failure and one haddelayed platelet engraftment on the TBI protocol and as a result 4additional patients were enrolled at that dose level. Of thoseadditional patients, no further graft toxicities were observed. Therewere five patient deaths but none attributable to the study drug.

The primary toxicity in all patients treated to date has been mucositis.This toxicity has been well managed in all patients, and no patientshave experienced any mucositis >grade 2. The addition of targetedradiotherapy has not resulted in any increased toxicity beyond whatwould be expected in the standard treatment population. Representativecase studies are described in Examples 12-14, below.

In order to achieve a complete response using protocol criteria, apatient must have a complete absence of any myeloma protein in theblood/urine and marrow post treatment. The patient must have normal bonemarrow with complete resolution of plasmocytomas and no increase in bonelesions. To meet international standards, this must be maintained for 6weeks. While response rates to a conventional high dose therapy varywidely, in general for previously treated patients, there is a range of5-25% CR rate.

Partial response is defined as sustained decrease in the production rateof the monoclonal serum protein to 25% or less of the pretreatment valuefor at least 2 months. Calculations consider the serum myeloma proteinconcentration, variations in catabolic rate with changing concentration,and changes in estimated plasma volume. Response requires a sustained 24hour urine Bence Jones protein excretion to less than 0.1 gm/day for atleast 2 measurements.

Based on the 40 patients that have response data and have beenmonitored, currently there is a 45% complete response rate, with anoverall response rate (CR and PR) of 55% on these protocols.

EXAMPLE 12

Treatment: 20 Gy ¹⁶⁶Ho-DOTMP, Melphalan 140 mg/m²

The patient, a 47 year-old male with an original diagnosis of multiplemyeloma, was administered a therapeutic dose of ¹⁶⁶Ho-DOTMP of 3875 mCiwhich was calculated to deliver 40 Gy to the marrow. Post ¹⁶⁶Ho-DOTMP,the patient received a dose of 140 mg/m² of melphalan (I.V.). Thepatients stem cells were reinfused three days after the melphalan andwere followed by G-CSF for ten days.

Nine days post stem cell transplant, the patient engrafted neutrophils(ANC >500), and fourteen days post transplant, the patient engraftedplatelets (>20,000). Twenty-eight days post transplant the patient wasin complete remission.

EXAMPLE 13

Treatment: 20 Gy ¹⁶⁶Ho-DOTMP, Melphalan 140 mg/m²+800 cGy Total BodyIrradiation

The patient, a 54 year-old male with an original diagnosis of free kappalight chain multiple myeloma and currently with primary refractorydisease, was administered 29.1 mCi (61.4 cGy of ¹⁶⁶Ho-DOTMP as adiagnostic dose. Based on dosimetry, a therapeutic dose of 1660 mCi(61.46 GBq) was calculated to deliver 20 Gy to the marrow. The actualtherapeutic dose injected was 1555 mCi. Three days post ¹⁶⁶Ho-DOTMP thepatient received a dose of 140 mg/m² of Melphalan (I.V.) for a totaldose of 269 mg. One day post Melphalan the patient received the first offour days of total body irradiation (TBI). The total dose of TBI was 800cGy fractionated into four doses of 200 cGy. The patient's stem cellswere reinfused after the last dose of TBI and were followed by G-CSF ata total dose of 480 mcg for ten days.

Ten days post stem cell transplant the patient engrafted neutrophils(absolute neutrophil count (ANC)>500), and nine days post transplant thepatient engrafted platelets (>20,000). One month post treatment thepatient was determined to have a complete remission.

EXAMPLE 14

Treatment: 20 Gy ¹⁶⁶Ho-DOTMP, Melphalan 140 mg/m²

The patient, a 59 year-old female with an original diagnosis of multiplemyeloma and currently diagnosed with primary refractory disease, waspremedicated with an antiemetic, IV Zofran at 8 mg Q8h, this wascontinued post injection for three days.

The patient was administered 29.4 mCi of ¹⁶⁶Ho-DOTMP as a diagnosticdose, based on dosimetry a therapeutic dose of 582 mCi was calculated todeliver 20 Gy to the marrow. The actual therapeutic dose injected was460 mCi. Six days post ¹⁶⁶Ho-DOTMP the patient received a dose of 140mg/m² of Melphalan (I.V.) for a total dose of 220 mg. The patient's stemcells were reinfused three days after the Melphalan and were followed byG-CSF at a total dose of 300 mcg for ten days.

Ten days post stem cell transplant the patient engrafted neutrophils(absolute neutrophil count (ANC)>500), and fourteen days post transplantthe patient engrafted platelets (>20,000). Five months post treatmentthe patient was determined to have a complete remission.

EXAMPLE 15

Stability of Metal Ligand Complexes With Stabilizer:

Samples of ¹⁶⁶Ho-DOTMP were prepared according to the procedure inExample 1 using ascorbic acid, 55 mg/mL, as the stabilizer. Identicalsamples were prepared without ascorbic acid. The solutions were analyzedfor radiochemical purity after 1 hour, 6 hours, 10 hours, 24 hours, and48 hours, using Instant Thin Layer Chromatography (ILTC), CationExchange Chromatography (CEC) and High Performance Liquid Chromatography(HPLC). As can be seen in the Table 3, the use of a radioprotectant(stabilizer) allowed the sample to maintain high radiochemical purityover samples without any stabilizer. TABLE 3 Time (hrs) 1 6 10 24 48ITLC Without stabilizer 99.2 98.1 97.5 97.6 96.5 With stabilizer 99.099.2 99.6 99.6 99.6 CEC Without stabilizer 99.0 97.8 97.8 97.2 97.1 Withstabilizer 98.4 99.0 99.6 98.5 98.7 HPLC Without stabilizer 100 95.494.9 85.8 With stabilizer 100 100 99.0 98.7

EXAMPLE 16

Biodistribution Study of ¹⁶⁶Ho-DOTMP in Rats

Sprague Dawley (S. D.) rats were injected intravenously (inj. i.v.) witha solution of ¹⁶⁶Ho-DOTMP (“Ho-DO”) containing ascorbic acid (asc) as astabilizer. The animals were sacrificed and organs excised and countedin a radioactive well counter after decay to appropriate levels. Bone(femur) samples were counted and converted to a total bone percentinjected dose using a factor of 25 times femur percent.

A second group of Sprague Dawley rats were injected intravenously with asolution of ¹⁶⁶Ho-DOTMP without having the stabilizer. The animals weresacrificed and organs excised and counted in a radioactive well counterafter decay to appropriate levels. Bone (femur) samples were counted andconverted to a total bone percent injected dose using a factor of 25times femur percent.

The results of this study show that the addition of the stabilizingagent, ascorbic acid, lowered the uptake of radiation by the non-targetorgans, while equivalent bone uptake was seen. In both control andstabilized preparations, high uptake and specificity for skeletaltargeting was shown.

Results are illustrated summarized in Tables 4-6 and in FIGS. 2-4. FIG.2 illustrates the data uptake base on the % injection dose. FIG. 3illustrates the data uptake base on the % injection dose per gram(mass).FIG. 4 illustrates the data uptake base on the tissue/blood ratio.Abbreviations: Blo=blood; Tai=tail; Lun=lung; Liv=liver; Spl=spleen;Sto=stomach; Kid=kidneys; Int=intestines; Bon=bone; SD=standarddeviation. TABLE 4 Percent Injection Dose/Gram Ho-DO only SD Ho-DO + ascSD Blood 0.02 0.00 0.01 0.00 Tail 0.51 0.02 0.95 0.36 Lung 0.04 0.010.03 0.00 Liver 0.31 0.03 0.03 0.01 Spleen 1.03 0.24 0.08 0.02 Stomach0.05 0.03 0.04 0.03 Kidney 0.38 0.04 0.25 0.02 Intestine 0.23 0.11 0.130.03 Bone 4.84 0.52 4.65 0.29

TABLE 5 Percent Injection Dose Ho-DO only SD Ho-DO + asc SD Blood 0.290.04 0.16 0.03 Tail 3.21 0.14 6.19 2.11 Lung 0.00 0.00 0.00 0.00 Liver2.40 0.23 0.24 0.05 Spleen 0.56 0.11 0.04 0.01 Stomach 0.20 0.13 0.210.17 Kidney 0.72 0.07 0.47 0.03 Intestine 3.81 1.88 2.23 0.56 Bone 50.053.85 49.58 2.12

TABLE 6 Tissue/Blood Ratio Ho-DO only SD Ho-DO + asc SD Blood 1.00 0.001.00 0.00 Tail 31.33 3.84 104.71 16.20 Lung 2.34 0.45 3.01 0.50 Liver18.90 2.11 3.40 0.97 Spleen 62.09 12.64 8.53 1.85 Stomach 3.24 2.04 5.014.44 Kidney 22.84 1.33 30.69 8.12 Intestine 13.80 7.66 14.60 2.73 Bone296.80 52.34 563.40 137.47

EXAMPLE 17

Treatment of breast cancer will be in conjunction with high-dosecombination chemotherapy regimens such as CTCb (STAMP V):Cyclophosphamide 1500 mg/m², Thiotepa 125 mg/m², Carboplatin 200 mg/m²administered intravenously over one or several days. Chemotherapeuticswill preferably be administered following the Ho-DOTMP but may be givenprior to or simultaneously.

EXAMPLE 18

Breast cancer, particularly metastatic breast cancer, will be treatedwith the present complexes, e.g., with ¹⁶⁶Ho-DOTMP in accord with thepresent method, employing the regimens listed on Table 7. TABLE 7 BreastCancer Regimens Regimens Chemotherapeutic Agent(s) Combination RegimensAC Doxorubicin 40-45 mg/m² i.v., day 1 WITH Cyclophosphamide 200 mg/m²PO, days 3-6 Repeat cycle every 21 days OR Cyclophosphamide 500 mg/m²i.v., day 1 Repeat cycle every 28 days CAF(FAC) Cyclophosphamide 600mg/m² i.v., day 1 Doxorubicin 60 mg/m² i.v., day 1 Fluorouracil 600mg/m² i.v., days 1, 8 Repeat cycle every 28 days OR Cyclophosphamide 500mg/m² i.v., day 1 Doxorubicin 50 mg/m² i.v., day 1 Fluorouracil 500mg/m² i.v., days 1 Repeat cycle every 21 days and day 8 (FAC) CFMCyclophosphamide 600 mg/m² i.v., day 1 (CNF, FNC) Fluorouracil 600 mg/m²i.v., day 1 Mitoxentrone 12 mg/m² i.v., day 1 Repeat cycle every 21 daysCMF Cyclophosphamide 100 mg/m² PO, days 1-14 or 600 mg/m²/i.v., days 1,8 Methotrexate 40 mg/m² i.v., days 1, 8 Fluorouracil 600 mg/m² i.v.,days 1,8 Repeat cycle every 28 days OR Cyclophosphamide 600 mg/m² i.v.,day 1 Methotrexate 40 mg/m² i.v., day 1 Fluorouracil 600 mg/m² i.v., day1 Repeat cycle every 21 days NFL Mitoxantrone 12 mg/m² i.v., day 1Fluorouracill 350 mg/m² i.v., days 1-3, after Leucovorin Leucovorin 300mg i.v., over 1 hour, days 1-3 OR Mitoxantrone 10 mg/m² i.v., day 1Fluorouracil 1,000 mg/m²/d Cl, days 1-3, after leucovorin Leucorvorin100 mg/m² i.v., over 15 minutes, days 1-3 Repeat cycle every 21 daysSequential Doxorubicin 75 mg/m² i.v., every 21 days, for 4 Dox-CFMcycles followed by 21- or 280 day CMF for 8 cycles VATH Vinblastine 4.5mg/m² i.v., day 1 Doxorubicin 4.5 mg/m² i.v., day 1 Thlotepa 12 mg/m²i.v., day 1 Fluoxymesterone 20 or 30 mg/d PO Repeat cycle every 21 daysVinorelbine Vinorelbine 25 mg/m² i.v., days 1, 8 Doxorubicin Doxorubicin50 mg/m² i.v., day 1 Repeat cycle every 21 days Single-Agent RegimensAnastrozole Anastrozole 1 mg/d PO Capecitabine Capecitabine 1,250 mg/m²PO bid, days 1-14 Repeat cycle every 21 days CFM Cyclophosphamide 600mg/m² i.v., day 1 (CNF, FNC) Fluorouracil 600 mg/m² i.v., day 1Mitoxentrone 12 mg/m² i.v., day 1 Repeat cycle every 21 days DocetaxelDocetaxel 60-100 mg/m² i.v, over 1 hour, every 21 days GemcitabineGemcitabine 725 mg/m² i.v, over 30 minutes weekly for 3 weeks, followedby 1 week rest Repeat cycle every 28 days Letrozole Letrozole 2.5 mg/dPO Megestrol Megestrol 40 mg PO bid Paciltaxel Paciltaxel 250 mg/m² i.v,over 3 or 24 hours every 21 days OR Paciltaxel 175 mg/m² i.v., over 3hours, every 21 days Tamoxifen Tamoxifen 10 or 20 mg twice daily or 20mg/d PO Toremifene citrate Toremifene citrate 60 mg/d PO VinorelbineVinorelbine 30 mg/m² i.v, every 7 days

EXAMPLE 19

Prostrate cancer, particularly metastatic prostrate cancer will betreated with the present complexes, e.g., with ¹⁶⁶Ho-DOTMP, in accordwith the present method, employing the regimens listed on Table 8. TABLE8 Prostrate Cancer Regimens Regimen Chemotherapeutic Agent(s)Combination Regimens Estramustine Estramustine 200 mg/m² PO, tid, days1-42 Vinblastine Vinblastine 4 mg/m² i.v., weekly for 6 weeks, begin day1 Repeat cycle every 8 weeks FL Flutamide 250 mg PO, tid WITH Leuprolideacetate 1 mg/d SQ OR Leuprolide acetate depot 7.5 mg IM, every 28 daysi.v., day 1 FZ Flutamide 250 mg PO, tid WITH Goserelin acetate 3.6 mgimplant SQ, every 28 days OR Goserelin acetate 10.8 mg implant SQ every12 weeks Begin regimen 2 months prior to radiotherapy MitoxantroneMitoxantrone 12 mg/m² i.v., day 1 Prednisone Prednisone 5 mg PO, bidRepeat cycle every 21 days No Known Acronym Bloatutamide 50 mg/d PO WITHLeuprolide acetate depot 7.5 mg IM, every 28 days OR Goserelin acetate3.6 mg implant SQ, every 28 days PE Paciltaxel 120 mg/m² by 96-hour i.v.infusion, days 1-4 Estramustine 600 mg/d PO, qd, 24 hours beforepaciltaxel Repeat cycle every 21 days Single Regimens EstramustineEstramustine 14 mg/kg/d PO, in 3 or 4 divided doses Goserelin Goserelinacetate implant 3.6 mg implant SQ 8 weeks before radiotherapy, followedby 28 days by 10.8 mg implant SQ, every 12 weeks Nilutamide Nilutamide300 mg PO, days 1-30, then 150 mg PO/d in combination with surgicalcastration; begin on same day or day after castration PrednisonePrednisone 5 mg PO, bid

EXAMPLE 20 Treatment of Multiple Myeloma

Multiple myeloma will be treated with the present complexes, e.g., with¹⁶⁶Ho-DOTMP, in accord with the present method, employing the regimenslisted on Table 9. TABLE 9 Multiple Myeloma Regimens. RegimenChemotherapeutic Agent(s) Combination Regimens M2 Vincristine 0.03 mg/kgi.v., day 1 Carmustine 0.5-1 mg/kg i.v., day 1 Cyclophosphamide 10 mg/kgi.v., day 1 Melphalan 0.25 mg/kg PO, days 1-4 OR Melphalan 0.1 mg/kg PO,days 1-7 or 1-10 Prednisone 1 mg/kg/d PO, days 1-7 Repeat cycle every35-42 days MP Melphalan 8-10 mg/m² PO, days 1-4 Prednisone 60 mg/m² PO,days 1-4 Repeat cycle every 28-42 days VBMCP Vincristine 1.2 mg/m² i.v.,day 1 Carmustine 20 mg/m² i.v., day 1 Melphalan 8 mg/m²PO, days 1-4Cyclophosphamide 400 mg/m² i.v., day 1 Prednisone 40 mg/m² PO, days 1-7all cycles, and 20 mg/m² PO, days 8-14 first 3 cycles only Repeat cycleevery 35 days Single-Agent Regimens Dexamethasone Dexamethasone 20 mg/m²PO, for 4 days beginning on days 1-4, 9-12 and 17-20 Repeat cycle every14 days Interferon alfa-2b Interferon alfa-2b 2 million units/m² SQ 3times a week for maintenance therapy in selected patients withsignificant response to initial chemotherapy treatment MelphalanMelphalan 90-140 mg/m² i.v. Administer one cycle

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

All patents, patent applications, and literature cited in thespecification are hereby incorporated by reference in their entirety. Inthe case of any inconsistencies, the present disclosure, including anydefinitions therein will prevail.

1. A therapeutic method for treating leukemia comprising: (a) hydratinga leukemia patient; (b) parenterally administering a dose of about400-4000 mCi 153Sm-EDTMP to said patient; (c) administering a dose ofmelphalan, bisulfan, cyclophosphamide or a combination thereof, to saidpatient; and (d) providing the patient with a stem cell transplant;wherein the patient is not subjected to total body irradiation inconjunction with the therapeutic method.
 2. The method of claim 1wherein the patient is refractory to treatment, or in relapse aftertreatment with chemotherapy and/or total body irradiation.
 3. The methodof claim 1 or 2 wherein about 460-1600 mCi 153Sm-EDTMP is delivered instep (c).
 4. The method of claim 1 or 2 wherein about 30-50 Gy ofradiation are delivered in the bone marrow of the patient.
 5. The methodof claim 1 wherein the leukemia is myeloid leukemia.
 6. A therapeuticmethod for treating a bone-associated cancer in a human patientcomprising: (a) parenterally administering a dose of about 400-4000 mCiof 153Sm-EDTMP; (b) administering a dose of a second chemotherapeuticagent to said patients, wherein steps (a) and/or (b) are effective tosuppress the bone marrow of a human patient; and (c) providing thepatient with bone marrow transplantation and/or restoration; wherein thepatient is not subjected to total body irradiation in conjunction withthe therapeutic method.
 7. The method of claim 6 wherein step (c) iscarried out while the bone marrow is suppressed by steps (a) and (b). 8.The method of claim 6 wherein the patient is refractory to treatment ofin relapse after treatment with chemotherapy and/or total bodyirradiation.
 9. The method of claim 6 wherein the patient is hydratedprior to, during and/or after step (a).
 10. The method of claim 6, 7 or8 wherein the bone associated cancer is prostrate cancer or breastcancer that has metastasized to the bone.
 11. The method of claim 6, 7or 8 wherein about 460-1600 mCi is administered in step (b).
 12. Themethod of claim 1 or 5 whereas the bone marrow transplantation orrestoration comprises bone marrow transplantation, stem celltransplantation and/or administration of a colony stimulating factor.13. The method of claim 6, 7, 8 and 9 wherein the dose of 153Sm-EDTMP isdelivered by intravenous infusion or injection in an aqueous vehiclecomprising an effective antiradiolytic amount of a pharmaceuticallyacceptable radioprotectant.
 14. The method of claim 13 wherein theradioprotectant is an ascorbate or gentistic acid.
 15. The method ofclaim 14 wherein the ascorbate is ascorbic acid at a concentration ofabout 35-75 mg/ml.
 16. The method of claim 6, 7, 8 or 9 wherein the dosedelivers about 30-40 Gy of radiation to the bone marrow of the patient.17. The method of claim 6, 7, 8 or 9 wherein the dose delivers about15-30 Gy of radiation to the bone marrow of the patient.
 18. The methodof claim 6, 7, 8 or 9 wherein about 140-200 mg/m2 of melphalan isadministered.